Mycobacterium tuberculosis produces chronic, asymptomatic infections in most people. This project seeks to define the mechanisms of this sustained infection-virulence determinants that enable the organism to survive inside human macrophages. The PI has been studying the interaction of epithelial cells with enteropathogenic E. coli (EPEC) for the last 8 years; methods established in the EPEC study will be used to identify and characterize M. tuberculosis gene sequences and their encoded products that mediate the organism's 1) entry into non-phagocytic and phagocytic mammalian cells, and 2) survival inside human macrophages. Preliminary studies have identified a 1,535-base-pair M. tuberculosis DNA fragment that confers HeLa cell invasion and macrophage intracellular survival to a non-pathogenic strain of Escherichia coli. Experiments will be conducted to isolate, purify, and characterize the product(s) of the cloned fragment. Because intracellular survival mechanisms are likely to be mediated by a variety of factors, we will clone additional sequences and their products by screening the M. tuberculosis genomic library in E. coli hosts using other selection systems, including cultured monocyte-macrophages, susceptible and relatively resistant strains of mice, and guinea pigs. This project will be conducted in collaboration with an investigator specializing animal models of mycobacterial infection. Identification of M. tuberculosis products associated with cell invasion and macrophage intracellular survival will contribute to the basic understanding of mycobacterial pathogenesis, and to the development of products that have potential therapeutic and preventative applications.